PROJECT SUMMARY The interaction between Hsp70s and cellular membranes is a new and largely uncharacterized function of these indispensable molecular chaperones. We hypothesize that the interaction between Hsp70s and lipids is a critical step for their membrane-associated functions, and that lipid-binding provides them with the necessary specificity to localize and function at different membranes during cellular stress and disease conditions like cancer. The interaction of Hsp70s with lipids suppresses tumor growth, induces cell death, activates the immune system, stabilizes membranes, and regulates nutrient recycling (microautophagy). This interaction depends on the lipid environment, is mediated by multiple types of molecular forces, and is altered by nucleotide binding. Furthermore, Hsp70s are differentiated with respect to their lipid-binding function. However, the conditions under which Hsp70s interact with lipids in human cells and the amino acid residues responsible for binding remain mostly unknown. To answer these two fundamental questions we propose two specific aims. First, we will determine the conditions that favor the interaction of HspA1A, the stress-inducible Hsp70 in humans, with lipids. For this task we will use several human cell lines, which will be subjected to different treatments that alter membrane lipid composition. The interaction of HspA1A with lipids will be assessed using pull-down assays, cellular imaging in the presence or absence of known lipid-binding proteins and fluorescent lipids, subcellular fractionation, and cell surface biotinylation. Furthermore, a targeted lipidomics approach will be used to authenticate HspA1A native lipid ligands. Second, we will identify and characterize the amino acids that mediate the HspA1A-lipid binding, and elucidate the molecular mechanism of this interaction. For this task, several amino acids, which will be predicted using computational techniques and observations from the literature, will be mutated. The mutational effect on the binding of recombinant HspA1A to lipids will be quantified using the liposome sedimentation method and Surface Plasmon Resonance spectroscopy. Additionally, the mutational effect on HspA1A function and stability will be determined by assessing alterations of the chaperone function. Finally, the mutational effect on the lipid-binding properties of HspA1A will be verified in human cells using fluorescently labeled HspA1A and a combination of pull-down assays, imaging, and subcellular fractionation. This proposal will provide fundamental knowledge that will allow us to test the effects of loss-of-function mutations in human cells and identify their physiological implications. If validated by these experiments, our lipid-binding specificity hypothesis will allow us to further elucidate this novel property of Hsp70s, which has critical associations with the cellular stress response, membrane biology, and disease conditions like cancer. Furthermore, this project will train multiple non-traditional and first-generation undergraduate and master level students, and will prepare them to enter companies, research labs, and advanced academic programs.